Signs of Doping or of Having Eaten Steak?

Pharmacy professor Maria Kristina Parr studies evidence of use of the muscle-building drug clenbuterol in athletes.

Jun 26, 2013

No other international sporting event has had such massive problems with doping as the Tour de France. Image Credit: Wikipedia.de / Wladyslaw

In the fight against doping in elite competitive sports, monitors have better and better tests at their disposal. But sometimes positive traces of a substance are not enough to provide definitive proof of abuse. A research group at the Institute of Pharmacy at Freie Universität, with funding from the World Anti-Doping Agency, is working to develop a test that aims to prove whether an athlete has doped using the muscle-building drug clenbuterol or whether the active ingredient has entered the athlete’s body through contaminated food.

Alberto Contador and Dimitrij Ovtcharov have something in common, although they could hardly be more different as athletes. Contador grew up near Madrid and has been a professional cyclist since 2003. He is now the celebrated winner of the three most important multi-stage national races in the cycling world (the Tour de France, Giro d'Italia, and Vuelta a España) and a global star worth millions. Ovtcharov, for his part, moved from Kiev to Germany with his parents, under a special German government program granting citizenship to certain groups from Eastern Europe who showed German descent. He has played in the Tischtennis-Bundesliga, the top professional table tennis league in Germany, since 2005, and is world-class in a sport that – at least in German public life – receives attention only during the Olympics, if then, and otherwise leads a shadowy existence.

But both have been accused of doping before sports tribunals – and they also have the same defense strategy. Monitors caught Contador in 2010, during the Tour de France, when his samples showed traces of clenbuterol, an asthma drug that also boosts fat burning and helps build muscle. Ovtcharov also tested positive that same year, when he was returning from a trip to China with the German national team. Both athletes have claimed in their defense that they must have eaten meat contaminated with the drug.

Because of its effects on muscle growth, clenbuterol is in fact sometimes used in animal feed, a practice that is illegal in the European Union. But while Ovtcharov was acquitted because he was able to provide evidence that he had eaten contaminated meat in China, and because a hair sample showed that he had not taken the drug for a longer period, Contador was hit hard. He retroactively lost all his winning titles since the 2010 Tour de France and was banned for two years.

So far, though, it has not been possible to prove definitively either that Ovtcharov is innocent or that Contador committed a deliberate doping violation – a situation that is unsatisfactory for all concerned. Maria Kristina Parr, a professor of pharmaceutical chemistry at the Institute of Pharmacy at Freie Universität, is currently working with her team to develop a test that, in the future, could reliably convict competitive athletes of deliberate clenbuterol abuse. “Clenbuterol is a highly effective medication, and the dosage is small, so the systems used to prove it must be very sensitive,” Parr says. Twenty picograms – 20 billionths of a gram – in a milliliter of urine is enough to give a positive result. “It’s as if you were to take an Olympic swimming pool, 50 by 25 meters and two meters deep, and dissolve a single sugar cube in it, then take a sample the size of a shot glass and then dilute that sample in another two liters of water,” says Parr.

With that in mind, the researcher’s approach to proving abuse lies not in the quantity of the substance that is proven to exist in the sample, but rather in its chemical structure. Clenbuterol comes in two forms. The two forms are identical in mass, composition, and molecular formula – but they differ in their three-dimensional molecular structure. “You might think of it as being like a left hand and a right hand,” Parr explains. “Both have five fingers and can grasp things, but on one hand, the thumb is on the left, while on the other, it is on the right.” And just like every person has one hand that works better than the other, one form of clenbuterol, (R)-clenbuterol, is more effective in the bodies of humans and animals than the other, (S)-clenbuterol. Both forms are also distributed and processed differently in the body.

In pharmaceutical practice, both forms of clenbuterol are used interchangeably because it would be much too expensive to separate the more effective form from the less effective one. “Our idea was to see whether it is possible to prove a changed relationship between (R)-clenbuterol and (S)-clenbuterol in the flesh of an animal,” Parr says. To find out, scientists working at the research department of the Dutch food monitoring and testing institute RIKILT – the partner in this research project – treated a calf with clenbuterol. What concentrations of the two forms of the active ingredient would be detected in the animal’s flesh?

To obtain the right measurement, the researchers had to develop a new method of separating the two forms. They decided to use chromatography, in which the substance to be analyzed is pumped through a “separating column.” At the end of this column, a steel cylinder approximately 20 centimeters long, the two forms of clenbuterol emerge separately, one at a time, and the respective quantity of the substance can be determined using a mass spectrometer.

That might sound simple, but it meant a huge amount of work for Parr and her team, since various fluids and parameters had to be tested. Two methods turned out to be promising: high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). Now further tests should show which of the two methods is better, and whether this research approach can be developed into a reliable doping test.

One initial result has given Parr confidence, at any rate: The group was able to show a ratio of one to 0.7 between ineffective and effective clenbuterol in the animal’s flesh. This shows that the animal’s body absorbed the two different forms in different concentrations. “Now the next step is to find out how clenbuterol behaves as a medication in the human body,” Parr says.